Figure 1
From: Calcium signalling in mammalian cell lines expressing wild type and mutant human α1-Antitrypsin
Characterisation of human alpha-1-antitrypsin (AAT) protein in CHO K1 cell lines expressing human wild type and disease variants of AAT. (a) Cartoon representation of the doxycycline inducible pTRE2hyg constructs encoding for human AAT proteins expression used in generating the stably AAT expressing CHO K1 cells line25. Single amino acid substitution from Glutamate (Glu) to Lysine (Lys) in the AAT sequence at position 342 resulted in the polymerogenic ZAAT (E342K) mutant (blue, 395 aa) that is retained in the ER. Frameshift mutation in the AAT sequence generating a premature stop codon at amino acid position 334 resulted in the NHK AAT mutant. Unlike the ZAAT, the NHK AAT mutant is cleared from the ER through the ER-associated degradation (ERAD) process. Human AAT protein expression in stably AAT expressing CHO K1 and control cells (non-AAT expressing) was evaluated by western blotting using an anti-antitrypsin 2G7 antibody that recognises both monomer and polymer forms of AAT and GAPDH as loading control. Arrows indicate the non-polymerogenic wild type MAAT and polymerogenic disease variant ZAAT proteins (~52 kDa)86 and the truncated form of the AAT protein, NHK AAT variant (~45 kDa)67. Full image of the blot is included in this paper as Supplemental Fig. 1. (b) Densitometry quantification of AAT detection in CHO K1 cells lines, expressed relative to the level of GAPDH. Data points plotted are means ± SEM. *P < 0.05; **P < 0.01 (n = 8 independent experiments). Controls are CHO K1 cells transfected with empty plasmids and otherwise treated in the same way as the AAT lines.
